C3 and C4 pathways

C3 and C4 pathways is explained in detail.

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It was once thought that all green plants fix CO2 through Calvin cycle only.

Now, we know that certain plants fix CO2 in a different photosynthetic mechanism called C4 pathway.

In this chapter, we will know more about this.

Hatch and Slack observed that 4C compounds such as oxaloaceticacid, malate and aspartate were the first formed compounds, when the leaves of sugarcane were exposed to 14CO2 for one second.

So, sugarcane is an example for C4 plant.

When the leaves of rice plant are exposed to 14CO2 3C compound called phosphoglyceric acid is formed.

So, rice plant is an example for C3 plant.

In C3 plants, photosynthesis occurs only in mesophyll cells.

We already learnt that photosynthesis has two types of reactions – light reactions and dark reactions (Calvin cycle).

In light reactions ATP and NADPH2 are produced and oxygen is released as a byproduct.

CO2 is reduced to carbohydrates by dark reactions.

In C3 plants both light reactions and dark reactions occur in mesophyll cells, whereas in C4 plants, the mechanism of photosynthesis requires two types of photosynthetic cells – mesophyll cells and bundle sheath cells.

The C4 plants contain dimorphic chloroplasts

i.e. chloroplasts in mesophyll cells are granal (with grana) whereas in bundle sheath chloroplasts are agranal (without grana).

The presence of two types of cells leads to segregation of photosynthetic work

i.e. light reactions and dark reactions separately.

Hatch-Slack pathway involves two carboxylation reactions.

C3 and C4 pathways

One takes place in chloroplasts of mesophyll cells and another in chloroplasts of bundle sheath cells.

1. The first step involves the carboxylation of phosphoenol pyruvic acid in the chloroplasts of mesophyll cells to form a 4C compound, oxaloacetic acid.

This reaction is catalysed by the enzyme phosphoenol pyruvate carboxylase

2. Oxaloacetic acid is converted into aspartic acid by the enzyme transaminase or it may be reduced to malic acid by NADP+ specific malate dehydrogenase.

3. Malic acid or aspartic acid formed in chloroplast of mesophyll cells is transferred to the chloroplasts of bundle sheath where it is decarboxylated to form CO2 and pyruvic acid in the presence of NADP+specific malic enzyme.

4. Now, second carboxylation occurs in chloroplasts of bundle sheath cells.

Ribulose bisphosphate accepts CO2 produced in step (3) in the presence of RuBP carboxylase and yields 3-phosphoglyceric acid.

Some of the 3-phosphoglyceric acid molecules are utilised to produce sucrose and starch, while remaining PGA molecules are used for the regeneration of RuBP.

5. The pyruvic acid produced in step (3) is transferred to the chloroplasts of mesophyll cells where it is phosphorylated to regenerate phosphoenolpyruvic acid .

This reaction is catalysed by pyruvate kinase in the presence of Mg 2+.

The AMP is phosphorylated by ATP in the presence of adenylate kinase to form 2 molecules of ADP.

C4 plants are photosynthetically more efficient than C3 plants, because the net requirement of ATP and NADPH2 for the fixation of one molecule of CO2 is considerably lower in C4 plants than in C3 plants.

Difference between C3 and C4 photosynthetic pathways

Diff b/w C3 and C4 pathways

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Other links 

Plant tissue culture – origin and techniques

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Plant physiology – photosynthesis and its significance

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Site of photosynthesis and Mechanism of photosynthesis

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Electron transport system and photophosphorylation types

---

Dark reaction

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Photorespiration or C2 cycle

---

Factors affecting photosynthesis

---

Test tube and funnel experiment, Ganong’s light screen experiment

---

Mode of nutrition – Autotrophic, Heterotrophic

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Chemosynthesis

---

Mechanism of Respiration – Glycolysis

---

Mechanism of Respiration – Oxidative decarboxylation , Krebs cycle

---

Mechanism of Respiration – Electron Transport Chain, Energy Yield

---

Ganong’s respiroscope, Pentose phosphate pathway

---

Anaerobic respiration, Respiratory quotient, Compensation point, Kuhne’s fermentation tube experiment

---

Plant growth and Measurement of plant growth

---

Phytohormones Auxins

---

Phytohormones Gibberellins

---

Phytohormones Cytokinin, Ethylene, Abscisic Acid, Growth Inhibitors – Physiological Effects

---

Photoperiodism and vernalization, Phytochromes and flowering

 

Dark Reaction

Dark reaction is expained in detail by the data below.

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The reactions that catalyze the reduction of CO2 to carbohydrates with the help of the ATP and NADPH2 generated by the light reactions are called the dark reaction.

The enzymatic reduction of CO2 by these reactions is also known as carbon fixation.

These reactions that result in CO2 fixation take place in a cyclic way and were discovered by Melvin Calvin.

Hence, the cycle is called Calvin cycle.

Fixation of carbondioxide in plants during photosynthesis occurs in three stages – fixation, reduction and regeneration of RuBP.

Dark reaction Carbon fixation 

The acceptor molecule of CO2 is a 5C compound called ribulose-1,5¬ bisphosphate (RuBP).

Fixation of a molecule of CO2 to RuBP is catalyzed by the enzyme RuBP carboxylase.

The resulting 6C compound is highly unstable and gets cleaved to form two molecules of 3C compounds called phosphoglyceric acid (PGA).

Dark reaction Reduction

The two molecules of PGA are further reduced to glyceraldehyde-3¬ phosphates in two steps.

First, two PGA molecules are converted to 1,3 -bisphosphoglyceric acids by the enzyme PGA kinase.

This reaction consumes two molecules of ATP in the ratio of one ATP for each molecule of 1,3-bisphosphoglyceric acid formed.

In the second step, the two molecules of 1,3-bisphosphoglyceric acid are reduced to glyceraldehyde-3-phosphates by the enzyme glyceraldehyde¬3-phosphate dehydrogenase with the help of the light generated reducing power NADPH2.

So, two molecules of NADPH2 will be consumed during this reaction.

To reduce one molecule of CO2 upto reduction two ATP and two NADPH2 are consumed.

Dark reaction Regeneration of RuBP

The glyceraldehyde 3-phosphate molecules are converted to RuBP through a series of reactions, which generate 4C, 6C and 7C phosphorylated compounds as intermediates.

For better and easy understanding of these reactions, a simplified scheme of Calvin cycle considering three CO2 molecules fixation reactions is shown below.

The reactions of regeneration of RuBP are as follows.

1. Some of the Glyceraldehyde 3-phosphate molecules are converted to dihydroxy acetone phosphates.

2. Glyceraldehyde 3-phosphate combines with dihydroxy acetone phosphate to form fructose1,6-bisphosphate.

3. Fructose 1,6-bisphosphate undergoes dephosphorylation to form fructose 6-phosphate.

4. Fructose 6-phosphate combines with glyceraldehyde 3-phosphate obtained from the fixation of second molecule of CO2 to form Ribose 5-phosphate (R5P) and Erythrose 4-phosphate (Er4P).

5. Erythrose 4-phosphate combines with DHAP obtained from the second CO2 fixation, to form sedoheptulose 1,7-bisphosphate.

6. Sedoheptulose 1,7-bisphosphate undergoes dephosphorylation to form sedoheptulose 7-phosphate.

7. Sedoheptulose 7-phosphate combines with glyceraldehyde 3-phosphate obtained by the third CO2 fixation, to form two molecules of 5C compounds – ribose 5-phosphate and xylulose 5-phosphate (Xy5P).

8. Ribose 5-phosphate and xylulose 5-phosphate molecules are transformed to ribulose 5-phosphate (Ru5P).

9. Ru5P molecules are then phosphorylated by ATP to form RuBP molecules, which again enter into the cycle of CO2 fixation.

In the above illustration, three CO2 molecules are fixed and the net gain is a 3C called DHAP.

These triose phosphate molecules combine to form hexose phosphates, which are used to form sucrose.

For every carbon fixation 3ATP and 2 NADPH2 are consumed

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For more information about Dark Reaction click here

Other links 

Plant tissue culture – origin and techniques

---

Plant physiology – photosynthesis and its significance

---

Site of photosynthesis and Mechanism of photosynthesis

---

Electron transport system and photophosphorylation types

---

 

C3 and C4 pathways

---

Photorespiration or C2 cycle

---

Factors affecting photosynthesis

---

Test tube and funnel experiment, Ganong’s light screen experiment

---

Mode of nutrition – Autotrophic, Heterotrophic

---

Chemosynthesis

---

Mechanism of Respiration – Glycolysis

---

Mechanism of Respiration – Oxidative decarboxylation , Krebs cycle

---

Mechanism of Respiration – Electron Transport Chain, Energy Yield

---

Ganong’s respiroscope, Pentose phosphate pathway

---

Anaerobic respiration, Respiratory quotient, Compensation point, Kuhne’s fermentation tube experiment

---

Plant growth and Measurement of plant growth

---

Phytohormones Auxins

---

Phytohormones Gibberellins

---

Phytohormones Cytokinin, Ethylene, Abscisic Acid, Growth Inhibitors – Physiological Effects

---

Photoperiodism and vernalization, Phytochromes and flowering

 

Cyclic photophosphorylation and non Cyclic photophosphorylation

Cyclic photophosphorylation and non Cyclic photophosphorylation

Electron transport system

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The light driven reactions of photosynthesis are referred to as electron transport chain.

When PS II absorbs photons of light, it is excited and the electrons are transported through electron transport chain of plastoquinone, cytochrome b6, cytochrome f and plastocyanin.

The electrons released from PS II phosphorylate ADP to ATP.

This process of ATP formation from ADP in the presence of light in chloroplast is called photophosphorylation.

Now, the PS II is in oxidised state.

It creates a potential to split water molecules to protons, electrons and oxygen.

This light dependent splitting of water molecules is called photolysis of water.

Manganese, calcium and chloride ions play prominent roles in the photolysis of water.

The electrons thus released are used in the reduction of PS II.

Similar to PS II, PS I is excited by absorbing photons of light and gets oxidised.

This oxidised state of the PS I draws electrons from PS II and gets reduced.

The electrons released to PS I are transported through electron transport chain of ferredoxin reducing substrate, ferredoxin and ferredoxin NADP reductase to reduce NADP+ to NADPH2.

Cyclic and noncyclic photophosphorylation

In chloroplasts, phosphorylation occurs in two ways – non cyclic photophosphorylation and cyclic photophosphorylation.

Non cyclic photophosphorylation

When the molecules in the PS I are excited the electrons are released. So, an electron deficiency or a hole is made in the PS I.

This electron is now transferred to ferredoxin to reduce NADP+.

When the molecules in the PS II get excited, electrons are released.

They are transferred to fill the hole in PS I through plastoquinone, cytochrome b6, cytochrome f and plastocyanin.

When the electron is transported between plastoquinone and cytochrome f, ADP is phosphorylated to ATP.

The ‘hole’ in the PS I has been filled by the electron from PS II.

Then the electrons are transferred from PS I to NADP+ for reduction.

Therefore, this electron transport is called noncyclic electron transport and the accompanying phosphorylation as noncyclic photophosphorylation.

The noncyclic electron transport takes place in the form of ‘Z’. Hence, it is also called Z-scheme.

Cyclic photophosphorylation

Under the conditions of

(i) PS I only remains active

(ii) photolysis of water does not take place

(iii) requirement of ATP is more and

(iv) nonavailability of NADP+ the cyclic photophosphorylation takes place.

When the molecule in the PS I is excited, the electrons are released.

The electrons are captured by ferredoxin through ferredoxin reducing substrate (FRS).

Due to non-availability of NADP+, electrons from ferredoxin fall back to the molecules of PS I through the electron carriers ¬ cytochrome b6, cytochrome f and plastocyanin.

These electron carriers facilitate the down hill transport of electrons from FRS to PS I.

During this transport of electrons, two phosphorylations take place – one between ferredoxin and cytochrome b6 and the other between cytochrome b6 and cytochrome f.

Thus, two ATP molecules are produced in this cycle.

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For more information about photophosphorylation click here

Other links 

Plant tissue culture – origin and techniques

---

Plant physiology – photosynthesis and its significance

---

Site of photosynthesis and Mechanism of photosynthesis

---

Dark reaction

---

C3 and C4 pathways

---

Photorespiration or C2 cycle

---

Factors affecting photosynthesis

---

Test tube and funnel experiment, Ganong’s light screen experiment

---

Mode of nutrition – Autotrophic, Heterotrophic

---

Chemosynthesis

---

Mechanism of Respiration – Glycolysis

---

Mechanism of Respiration – Oxidative decarboxylation , Krebs cycle

---

Mechanism of Respiration – Electron Transport Chain, Energy Yield

---

Ganong’s respiroscope, Pentose phosphate pathway

---

Anaerobic respiration, Respiratory quotient, Compensation point, Kuhne’s fermentation tube experiment

---

Plant growth and Measurement of plant growth

---

Phytohormones Auxins

---

Phytohormones Gibberellins

---

Phytohormones Cytokinin, Ethylene, Abscisic Acid, Growth Inhibitors – Physiological Effects

---

Photoperiodism and vernalization, Phytochromes and flowering

 

Site of photosynthesis and Mechanism of photosynthesis

Site of photosynthesis and Mechanism of photosynthesis

Site of photosynthesis

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Chloroplasts are the actual sites for photosynthesis.

All green parts of a plant are involved in photosynthesis.

Leaves are the most important organs of photosynthesis.

In xerophytes like Opuntia, the stem is green and it performs photosynthesis.

Over half a million chloroplasts are present in one square millimetre of a leaf.

It measures about 4 to 6 micron.

A typical chloroplast of higher plants is discoid shaped.

It is a double membrane bound organelle containing chlorophyll, carotenoid,

xanthophyll, cytochrome, DNA, RNA, manganese, etc.

Chloroplasts are generally considerably larger than mitochondria.

The space enclosed by the envelope is filled with matrix called stroma.

In the stroma, many grana are embedded.

In each granum, several disc shaped lamellae are found.

These disc shaped structures are called thylakoids.

They resemble a stack of coins.

This structure is known granum.

Generally a chloroplast contains 40 to 60 grana.

The photosynthetic pigments are found in grana.

The stroma contains circular DNA, RNA and enzymes for starch synthesis.

Photochemical and biosynthetic phases

The pigments involved in photosynthesis are called photosynthetic pigments.

They are chlorophyll ‘a’, chlorophyll ‘b’, carotenoids, xanthophyll and phycobilins.

Magnesium is an essential component for the formation of chlorophyll.

Chlorophyll ‘a’ is a universal pigment present in the plants in which water is one of the raw materials for photosynthesis.

Chlorophylls are highly efficient in absorbing solar energy and they are directly linked to photosynthetic electron transport.

Photosynthetic pigments other than chlorophyll ‘a’ are generally called accessory pigments

eg. chlorophyll ‘b’, carotenoids and xanthophyll, whereas chlorophyll ‘a’ is regarded as primary pigment.

Photosynthetic pigments occur in the granum.

They constitute the pigment system called photosystem.

About 250 to 400 pigment molecules are present in a photosystem.

Two types of photosystems are found in the granum.

Photosystem I (PS I) has less accessory pigments and more chlorophyll ‘a’, while photosystem II (PS II) has more accessory pigments and less chlorophyll ‘a’.

The primary function of photosystems is to trap light energy and converts it to chemical energy.

The energy absorbed by accessory pigments is transferred to the chlorophyll ‘a’.

The granal lamella where the photosynthetic pigments are aggregated to perform photosynthetic activities is called active centre.

Mechanism of photosynthesis

The overall reaction of photosynthesis can be written as follows.

The reactions of photosynthesis can be grouped into two – light reactions and dark reactions.

The reactions involving pigments, solar energy and water that produce ATP and NADPH2 are called light reactions.

The photosynthetic reactions in which CO2 is reduced to carbohydrates making use of ATP and NADPH2 generated by light reactions are collectively called dark reactions.

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For more details about Mechanism of photosynthesis click here

Other links 

Plant tissue culture – origin and techniques

---

Plant physiology – photosynthesis and its significance

---

Electron transport system and photophosphorylation types

---

Dark reaction

---

C3 and C4 pathways

---

Photorespiration or C2 cycle

---

Factors affecting photosynthesis

---

Test tube and funnel experiment, Ganong’s light screen experiment

---

Mode of nutrition – Autotrophic, Heterotrophic

---

Chemosynthesis

---

Mechanism of Respiration – Glycolysis

---

Mechanism of Respiration – Oxidative decarboxylation , Krebs cycle

---

Mechanism of Respiration – Electron Transport Chain, Energy Yield

---

Ganong’s respiroscope, Pentose phosphate pathway

---

Anaerobic respiration, Respiratory quotient, Compensation point, Kuhne’s fermentation tube experiment

---

Plant growth and Measurement of plant growth

---

Phytohormones Auxins

---

Phytohormones Gibberellins

---

Phytohormones Cytokinin, Ethylene, Abscisic Acid, Growth Inhibitors – Physiological Effects

---

Photoperiodism and vernalization, Phytochromes and flowering

 

Plant physiology – photosynthesis and its significance

Plant physiology – photosynthesis and its significance

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Plant physiology is the branch of biological science, which deals with the functioning, and interrelationships of cells, tissues and organs of plants.

Green plants have the capacity of harvesting light energy for life energy, making use of inorganic raw materials.

Most of the living organisms including man depend upon this energy rich compounds of plants.

Plants not only provide food but also supply required oxygen for breathing.

Besides synthesizing organic compounds, plants carry other natural phenomena of living organisms such as respiration, growth and development.

In this chapter, we study these natural phenomena operating in plants.

Though the plants do not have respiratory, circulatory and digestive systems like animals, all these functions are carried out at cellular level.

Photosynthesis

Photosynthesis literally means ‘synthesis with the help of light’.

It is the process that gives life to all living beings.

The plants convert light energy into life energy.

It is the only biological process that makes use of sun’s light energy for driving the life machinery.

Hence, photosynthesis is regarded as ‘leader’ of all processes both biological and abiological.

It is the most fundamental of all biochemical reactions by which plants synthesize organic compounds in the chloroplast from carbondioxide and water with the help of sunlight.

It is an oxidation–reduction reaction between water and carbondioxide.

Significance of photosynthesis

• Photosynthesis is a source of all our food and fuel. It is the only biological process that acts as the driving vital force for the whole animal kingdom and for the non-photosynthetic organism.
• It drives all other processes of biological and abiological world, it is responsible for the growth and sustenance of our biosphere.
• Photosynthesis provides organic substances, which are used in the production of fats, proteins, nucleoproteins, pigments, enzymes, vitamins, cellulose, organic acids, etc. Some of them become structural parts of the organisms.
• It makes use of simple raw materials such as CO2, H2O and inexhaustible light energy for the synthesis of energetic organic compounds.
• It is significant because it provides energy in terms of fossil fuels like coal and petrol obtained from plants, which lived millions and millions of years ago.
• Plants, from great trees to microscopic algae, are engaged in converting light energy into chemical energy, while man with all his knowledge in chemistry and physics cannot imitate them.

History of photosynthesis

• 320 BC Ancient Indians believed that plants fed from their feet ¬ Padapa, refers to a plant which drinks from the feet.
• 1727 Stephen Hales recognised the importance of light and air in the nourishment of plants.
• 1779 Jan Ingen-Housz discovered that the green parts of the plant purify the polluted air in the presence of light.
• 1782 Senebier showed that as the concentration of CO2 increases, the rate of O2 evolution also increases.
• 1845 Von Mayer recognised that green plants convert solar energy into chemical energy of organic matter.
• 1845 Liebig pointed out that the organic matter was derived from CO2 and water.
• 1920 Warburg introduced the unicellular green alga Chlorella as a suitable material to study photosynthesis.
• 1932 Emerson and Arnold showed that the existence of light and dark reactions in photosynthesis.
• 1937 Hill demostrated photolysis of water by isolated chloroplasts in the presence of suitable electron acceptor.
• 1941 Ruben and Kamen used 18O2 to show that O2 comes from water in photosynthesis.
• 1954 Arnon, Allen and Whatley used 14CO2 to show fixation of CO2 by isolated chloroplasts.
• 1954 Calvin traced the path of carbon in photosynthesis and gave
  C3 cycle (Calvin cycle) and was awarded Noble prize in 1960.
• 1965 Hatch and Slack reported the C4 pathway for CO2 fixation
  in certain tropical grasses.

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For more information about photosynthesis click here

Other links 

Plant tissue culture – origin and techniques

---

 

Site of photosynthesis and Mechanism of photosynthesis

---

Electron transport system and photophosphorylation types

---

Dark reaction

---

C3 and C4 pathways

---

Photorespiration or C2 cycle

---

Factors affecting photosynthesis

---

Test tube and funnel experiment, Ganong’s light screen experiment

---

Mode of nutrition – Autotrophic, Heterotrophic

---

Chemosynthesis

---

Mechanism of Respiration – Glycolysis

---

Mechanism of Respiration – Oxidative decarboxylation , Krebs cycle

---

Mechanism of Respiration – Electron Transport Chain, Energy Yield

---

Ganong’s respiroscope, Pentose phosphate pathway

---

Anaerobic respiration, Respiratory quotient, Compensation point, Kuhne’s fermentation tube experiment

---

Plant growth and Measurement of plant growth

---

Phytohormones Auxins

---

Phytohormones Gibberellins

---

Phytohormones Cytokinin, Ethylene, Abscisic Acid, Growth Inhibitors – Physiological Effects

---

Photoperiodism and vernalization, Phytochromes and flowering

 

Single cell protein – SCP

Single cell protein – SCP and its uses

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Microorganisms have been widely used for preparation of a variety of fermented foods. Eg. cheese, butter, idlis, etc., in addition, some microorganisms have long been used as human food, eg. the blue green alga Spirulina, and the fungi commonly known as mushrooms.

More recently, efforts have been made to produce microbial biomass using low-cost substrates and use as a supplemental food for human consumption or used as feed for animals.

Cells from a variety of micro-organisms, viz., bacteria, yeasts, filamentous fungi and algae used as food or feed are called single cell protein (SCP).

The term ‘single cell protein’ was coined in 1966.

The dried cells of microorganisms used as food or feed for animals and they are collectively known as Microbial proteins.

This term was replaced by a new term ‘single cell protein’.

The isolated protein or the total cell material is called the single cell protein – SCP.

In view of the insufficient world food supply and the high protein content of microbial cells, the use of biomass produced in the fermentor (special sterilized vessel) or bio-reactor would be ideal supplement for conventional food.

Single cell protein is of great nutritional value because of its high protein, vitamin and lipid content and for its essential amino acids. In many countries,

however people hesitate to use Single cell protein SCP as a major food source because of the following

• The high nucleic acid content (4 to 6 per cent in algae, 6 to10 per cent in yeast of single cell protein – SCP) can cause health problems like uric acid formation, kidney stones and rheumatism in human beings.
• Toxic or carcinogenic (cancer causing) substances absorbed from the microbial growth substrate may be present.
• Slow digestion of microbial cell in the digestive tract may cause vomiting, indigestion and allergic reaction.
• High cost of production will also be a deciding factor in determining the ultimate place of single cell protein – SCP in the human or animal diet.
• The following substrates are being studied for single cell protein – SCP production: alkanes, methane, methanol, cellulose, carbohydrates and waste materials.
• Natural sources like wood chips, rice husk, cane and beet molasses, peas and coffee industrial waste from which cellulose is obtained and are used for the production of SCP.
• Large scale cultivation of yeast on molasses is widely used in the manufacture of Baker’s yeast containing mycoproteins which is used in the SCP production.
• Domestic sewage is not suitable for large scale SCP production. But it is more important for methane production. The industrial wastewater from cellulose processing, coffee and starch production, and food processing have been used for SCP production.

Organisms used for Single cell protein SCP production

Algae Chlorella, Spirulina and Chlamydomonas.

Fungi Saccharomyces cereviseae, Volvoriella and Agaricus campestris

Bacteria Pseudomonas and Alkaligenes

Uses of Single cell protein SCP

1. It is a rich source of protein (60 to 72 per cent), vitamins, amino acids, minerals and crude fibres.
2. It is a popular health food. Nowadays, Spirulina tablets are prescribed as enriched vitamin for most people.
3. It provides valuable protein-rich supplement in human diet.
4. It lowers blood sugar level of diabetics due to the presence of gamma. Linolenic acid and prevents the accumulation of cholesterol in human body.

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For more information about Single cell protein SCP click here

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Recombinant DNA technology

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Gene transfer in plants, Cutting of DNA, Advantages of recombinant DNA

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Transgenic plants – Herbicide resistance in transgenic plants

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Practical application of genetic transformation

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Plant tissue culture – origin and techniques

---

Basic techniques of plant tissue culture

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Status of tissue culture technology in India And application

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Protoplast fusion and Practical applications

---

 

Protoplast fusion and Practical applications

Protoplast fusion and Practical applications

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Protoplasts are cells without a cell wall but bound by a plasma membrane.

The isolated protoplasts are totipotent.

Because of this unique property, plant protoplasts play a vital role in the field of genetic engineering.

Protoplast technology includes the isolation, culture and fusion of higher plant protoplasts leading to the production of entire plants.

You will be studying the method of isolation and fusion of protoplast in this chapter.

A hybrid produced by fusion of somatic cells of two varieties (or) species is called somatic hybrid.

This process of producing somatic hybrids is known as somatic hybridization.

The first step in somatic hybridization is the isolation of protoplast.
Isolation of protoplast

Protoplast can be isolated from a variety of plant tissues using either mechanical (or) enzymatic methods.

Mechanical method

In this method, cells are kept in a suitable plasmolyticum (protoplast shrink away from cell wall in a plasmolysed cell) and cut with a fine knife, so that protoplasts are released from cells through the opening of the cell wall.

This method gives poor yield of protoplast and it is being rarely used.

Enzymatic method

Leaves from a 10 week old plant are sterilized with 70 per cent alcohol and then treating them with 2 per cent solution of sodium hypochlorite for 20 to 30 minutes.

The leaves are then washed with sterile water and subsequent procedures are done under aseptic conditions (using laminar air flow chamber).

The lower epidermis of the leaf is peeled off and the leaf is cut into small fragments.

From the peeled leaf segments, the protoplasts are isolated.

For isolation of protoplast, peeled leaf segments are placed with their lower surface downwards in a petridishes containing the enzyme mixture, which consists of 0.5 per cent macerozyme, 2 per cent cellulase in 13 per cent sorbitol or mannitol at pH 5.4.

Finally the protoplasts are released and are kept in the isotonic solution.

Protoplasmic fusion

Protoplast fusion facilitates mixing of two genomes and could be exploited in crosses which are not possible by conventional techniques due to incompatibility.

Even though transfer of a single gene from one plant to another is desirable and protoplast fusion facilitates easy monitoring of cell genetic changes.

Proto plast fusion could be spontaneous during isolation of protoplast or it can be induced by mechanical, chemical and physical means.

The isolated protoplasts are kept in isotonic solution (mannitol and enzyme mixture) to prevent damage.

The isolated parent protoplasts are fused with a fusogenic agent like Polyethylene glycol (PEG).

It is followed by nuclear fusion and results in a somatic hybrid.

The somatic hybrids are allowed to grow in the same culture medium.

The fused protoplast are then induced to regenerate the cell wall by transferring it into a suitable medium.

This is followed by callus formatiom which leads to regeneration and organization of tissues.

Practical applications of protoplasmic fusion

Due to the existence of incompatibility prevailing between different species, protoplasmic fusion greatly compensates for interspecific hybridization.

Somatic hybrids between rice and carrot were produced only through the process of protoplasmic fusion.

Somatic hybrids may be used for gene transfer, transfer of cytoplasm and production of useful allopolyploids.

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For more details about Protoplast fusion Click Here

Other links 

Recombinant DNA technology

---

Gene transfer in plants, Cutting of DNA, Advantages of recombinant DNA

---

Transgenic plants – Herbicide resistance in transgenic plants

---

Practical application of genetic transformation

---

Plant tissue culture – origin and techniques

---

Basic techniques of plant tissue culture

---

Status of tissue culture technology in India And application

---

 

Single cell protein and its uses

Status of tissue culture technology in India & Applications of plant tissue culture

Status of tissue culture technology in India And Applications of plant tissue culture

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India is reported to have one of the largest groups of tissue culture scientists in the world.

Most of the research is directed towards the development of improved plants for agriculture, horticulture and forestry using tissue culture methods.

The Department of Biotechnology (DBT), Government of India, New Delhi is playing a vital role in promoting research in the area of plant tissue culture.

Several laboratories are being supported by providing funds for development of tissue culture technology for the improvement of crop plants.

The important Biotechnology centres for tissue culture technology in India are

i. Indian Agricultural Research Institute (IARI), New Delhi.
ii. Bhaba Atomic Research Centre (BARC), Mumbai.
iii. Central Institute of Medicinal and Aromatic plants (CIMAP), Lucknow.
iv. Dr.M.S. Swaminathan Research Institute (MSSRI), Chennai.

Applications of plant tissue culture

• Several commercial establishments now routinely use micropropagation for different foliage and ornamental plants.
• Through tissue culture methods using bud proliferation and multiple shoot formation, ornamental plants are produced in large numbers.
• Virus free germplasm are produced through apical meristem culture eg. banana.
• Artificial synthetic seeds are produced through somatic embryogenesis.
• Plant tissue culture is an important technique for the production of secondary metabolites in large quantities.
• Tissue culture helps in induction of haploidy in anther culture ie. useful for mutation breeding, triploidy through endosperm culture for inducing parthenocarpic fruits and polyploidy for increase in biomass or yield.
• Embryo culture technique is applied to overcome embryo abortion, seed dormancy and self-sterility in seeds.
• In recent years, plant tissue culture methods are employed in plants for the introduction of foreign gene into plant cells through DNA coated microparticles and delivering these particles into a host cell by using a gene gun.
• Protoplasmic fusion encourages genomes of incompatible crops to come together to form somatic hybrids.
• Plant tissue culture is applied in the area of plant physiological and biochemical research to study the cell cycle, metabolism in cells, nutritional, morphogenetical and developmental studies in plants.
• By plant tissue culture techniques, a plant cell of potato and tomato were brought together through protoplasmic fusion and the hybrid cell was made to develop into a pomato plant. In pomato, the stem bears the tubers and the branches produced tomatoes.

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Recombinant DNA technology

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Gene transfer in plants, Cutting of DNA, Advantages of recombinant DNA

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Transgenic plants – Herbicide resistance in transgenic plants

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Practical application of genetic transformation

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Plant tissue culture – origin and techniques

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Basic techniques of plant tissue culture

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Protoplast fusion and Practical applications

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Single cell protein and its uses

Plant tissue culture and its application

Plant tissue culture – origin and techniques

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Plant tissue culture

Growing the plant cells, tissues and organs on a artificial, synthetic medium under controlled conditions is called plant tissue cultures.

Plant tissue culture has become a major thrust area in plant biotechnology.

Concept

The basic concept of plant tissue cultures is totipotency, differentiation, dedifferentiation and redifferentiation.

Totipotency

The inherent potential of any living plant cell to develop into entire organism is called totipotency. This is unique to plant cells.

Differentiation

The meristematic tissue is differentiated into simple or complex tissues.

Dedifferentiation

Reversion of mature tissue into meristematic state leading to the formation of callus is called dedifferentiation.

Redifferentiation

The ability of the callus to develop into shoot or root or embryoid.

The origin and development of plant tissue culture

The beginning of plant tissue culture was made as early as 1898, when a German Botanist G. Haberlandt successfully cultured individual plant cells, isolated from different tissues.

But only during 1934 to 1939, a foundation of plant tissue cultures was laid down by three scientists (Gauthret, White and Nobecourt) due to discovery of plant growth regulators such as auxins and vitamins.

During next twenty years (1940 to 1960) a variety of growth regulator such as cytokinins were identified for their effect on cell division, growth and differentiation.

After 1960, in vitro culture of plant cells, tissues and organs was reasonably well developed.

Research in this area was initiated in early 1960s by Prof. P. Maheshwari and Prof. S. Narayanaswamy at the Department of Botany, University of Delhi in India.

Consequently, media and culture techniques for a variety of plant materials became known, which are now extensively utilised in all areas of plant improvement programmes.

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For more details about plant tissue culture click here

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Recombinant DNA technology

---

Gene transfer in plants, Cutting of DNA, Advantages of recombinant DNA

---

Transgenic plants – Herbicide resistance in transgenic plants

---

Practical application of genetic transformation

---

 

Basic techniques of plant tissue culture

---

Status of tissue culture technology in India And application

---

Protoplast fusion and Practical applications

---

Single cell protein and its uses

Practical application of genetic transformation

Practical application of genetic transformation

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By genetic manipulation, it is possible to obtain plants with insecticidal property.

Thus, application of chemical pesticides to the crop plants is reduced.

Genetic manipulation is also carried out in crops for making desirable storage proteins, vitamins, amino acids and for the suppression of antinutritional protein synthesis.

Plants are made to produce large amount of secondary metabolites having high commercial value.

Losses during storage and transport of some crops can be as high as 80 per cent.

This is mainly due to biological activities – bruising, heat and cold damage in soft fruits and vegetables.

In tomato the enzyme polygalactronase breaks down cell wall constituents, thus leading to softening of the fruit during ripening.

By inhibiting the polygalactronase by antisense genes the tomato can remain dormant fresh until mature and be transported in a firm solid state.

Antisense RNA is a RNA molecule capable of controlling and expression of particular enzymes which are involved in ripening processes.

Genetic manipulation of flower and leaf colour, abundance of flowers, perfume and shape are now the major targets for decorative plant industries.

Transgenic microbes

The genetically engineered micro-organisms are being used for the commercial production of some non-microbial products such as insulin, interferon, human growth hormone and viral vaccines.

Use of genetically engineered bacterial strain

In 1979, for the first time Anand Mohan Chakrabarty, an Indian born American scientist developed a strain of Pseudomonas putida that contained a hybrid plasmid derived by combining parts of CAM and OCT.

CAM and OCT are the plasmids which contain the genes responsible for the decomposition of the hydrocarbons like camphor and octane respectively present in the oil.

This strain could grew rapidly on crude oil because it was capable of metabolizing hydrocarbons more efficiently.

The bacterial strain called the superbug was produced on a large scale in laboratory, mixed with straw and dried.

When the straw was spread over oil slicks, the straw soaked up the oil and bacteria broke up the oil into non-polluting and harmless products.

In this way, pollution of land and water due to the oil slicks can be remedied and the phenomenon is called bioremediation.

It is defined as the use of living microorganisms to degrade environmental pollutants or prevent pollution.

The contaminated sites are restored and future pollution is prevented.

Benefits from release of genetically modified microorganisms into the environment

Protection of environment – Bioremediation of polluted environment. 0 Microorganisms producing enzymes for food industry.

Microorganisms with improved efficiency of fermentation.

Improved microorganisms for milk industry.

Microorganisms as live attenuated vaccines for health care.

Increasing efficiency of plant nutrition, pest control (safe biopesticides), protection of plants from climatic stress and protection of plants from tumour formation and disease.

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For more details about application of genetic transformation click here

Other links 

Recombinant DNA technology

---

Gene transfer in plants, Cutting of DNA, Advantages of recombinant DNA

---

Transgenic plants – Herbicide resistance in transgenic plants

---

 

Plant tissue culture – origin and techniques

---

Basic techniques of plant tissue culture

---

Status of tissue culture technology in India And application

---

Protoplast fusion and Practical applications

---

Single cell protein and its uses